onpg beta lactase

7/26/2019 ONPG Beta Lactase

1/7

Effects of Carbohydrates on the oNPG Converting Activity ofGalactosidasesAnja Warmerdam, Jue Wang, Remko M. Boom, and Anja E. M. Janssen*

Food Process Engineering Group, Wageningen University, Bomenweg 2, 6703 HD, Wageningen, The Netherlands

*S Supporting Information

ABSTRACT: The effects of high concentrations of carbohydrates on the o-nitrophenyl -D-galactopyranoside (oNPG)converting activity of-galactosidase fromBacillus circulansare studied to get a better understanding of the enzyme behavior inconcentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. Thecomponents that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70 000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding ofthe galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem tocause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on

the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactantson -galactosidases from B. circulans, A. oryzae, and K. lactis are compared.

KEYWORDS: galacto-oligosaccharides, GOS, -galactosidase, Bacillus circulans, enzyme activity, carbohydrates, crowding,galactosyl transfer, transgalactosylation, Aspergillus oryzae, Kluyveromyces lactis

INTRODUCTION

Galacto-oligosaccharides (GOS) are widely recognized asprebiotics because they are indigestible in the human intestineand have a positive effect on the microora in the colon.1,2

They are commonlyadded to infant nutrition formula and toyogurts and drinks.2,3

GOS are typically produced from lactose via enzymatic

synthesis with -galactosidases.

1,35

A-galactosidase prepara-tion fromBacillus circulans, called Biolacta N5, is known for itshigh transgalactosylation activity compared to other -galactosidases.6,7 It produces relatively large amounts ofoligosaccharides with a higher degree of polymerizationcompared to -galactosidases from Aspergillus oryzae and

Kluyveromyces lactis for example.6,8 High GOS yields areobtained especially at high initial lactose concentrations.6,9

Reaction rates, equilibria, and mechanisms of biochemicalreactions are usually investigated in diluted systems. In order to

better understand lactose conversion in concentrated systems,where large amounts and many different types of reactants andproducts are present, it is essential to study the effect of thesecarbohydrates on GOS synthesis. Each of these reactants and

products may have its in

uence on GOS production: theymight be converted into other products or might inhibit thereaction.5 These reactants and products inuence the reaction,especially at high concentrations, and thus co-determine the

yield of GOS.Another phenomenon that mightplay a role in concentrated

systems is molecular crowding.10,11 The high concentration ofmolecules physically excludes some of the volume for othermolecules. The excluded volume is the volume in a system thatis occupied by molecules and cannot be occupied by the centerof other molecules. This means that the effective concentrationof components can be much larger than the actualconcentration. Molecular crowding can affect the reaction

rate, the reaction equilibrium, the enzyme activity, and thestability and can cause diffusion limitation.1214 Molecularcrowding has been mainly studied for a better understanding of

biochemical reactions in a cellular environment, but it is alsorelevant for enzymatic reactions under concentrated conditions.

The aim of this study is to investigate the effect of inert andreacting carbohydrates on the behavior of Biolacta N5 inconcentrated systems. The effects of broad concentration

ranges of several inert components (sarcosine and dextrans)and of reacting carbohydrates on the behavior of -galactosidase from B. circulans during oNPG conversion wereinvestigated. Because of the competitive effect between oNPGand other reactants (e.g., lactose), using the combination ofoNPG with these reactants will give more insight in themechanism than only using these reactants without oNPG.

The addition of reactants to theoNPG assay was investigatedfor-galactosidases from both A. oryzaeand K. lactis, to obtaina better mechanistic understanding of the activity of various -galactosidases and to investigate the reason of the high GOS

yields when using Biolacta N5.

MATERIALS AND METHODS

Materials.Lactose monohydrate (Lactochem), Vivinal GOS, and a-galactosidase preparation fromB. circulanscalled Biolacta N5 (DaiwaKasei K. K., Japan) were gifts from FrieslandCampina (Beilen, TheNetherlands). Biolacta N5was previously found to have a total proteincontent of 19 3%.15 In all calculations, the total enzymeconcentration was assumed to be equal to the total proteinconcentration, because the actual enzyme concentration is not known.

Received: February 22, 2013Revised: May 31, 2013Accepted: June 2, 2013Published: June 3, 2013

Article

pubs.acs.org/JAFC

2013 American Chemical Society 6458 dx.doi.org/10.1021/jf4008554|J. Agric. Food Chem.2013, 61, 64586464
http://localhost/var/www/apps/conversion/tmp/scratch_2/pubs.acs.org/JAFChttp://localhost/var/www/apps/conversion/tmp/scratch_2/pubs.acs.org/JAFC


2/7

-Galactosidase from A. oryzae (Lactase L017P) was a gift fromBiocatalysts (Nantgarw, UK). -Galactosidase from Kluyveromyceslactis (Lactozyme) was purchased from Sigma-Aldrich (Steinheim,

Germany). The enzyme concentration was calculated based on thetotal mass of solid and liquid taken from the preparation forA. oryzaeandK. lactis, respectively.

D-(+)-Galactose, D-(+)-glucose, sucrose, D-(+)-trehalose dihydrate,D-(+)-raffinose pentahydrate, sarcosine (N-methylglycine), dextran-6000 and -70 000 (from Leuconostocspp .), maltotriose, maltotetraose,maltopentaose, maltohexaose, maltoheptaose, sulfuric acid, sodiumhydroxide, o-nitrophenyl -D-galactopyranoside (oNPG), and o-nitrophenol (oNP) were purchased from Sigma-Aldrich (Steinheim,Germany). Sodium carbonate, citric acid monohydrate, and disodiumhydrogen phosphate were purchased from Merck (Darmstadt,Germany).

McIlvaines buffer was prepared by adding together 0.1 M citric acidand 0.2 M disodium hydrogen phosphate in the right ratio to achieve apH of 6.0 or 8.0.

Effect of Carbohydrates on Activity. The enzyme activity

measurements were adapted from Nakanishi et al.7

A stock solution ofoNPG in buffer with a concentration of 0.25% (w/w) was prepared. Inaddition, solutions of galactose, glucose, lactose, sucrose, trehalose,raffinose, sarcosine, dextran-6000, and dextran-70 000 in buffer withvarying concentrations were prepared. Stock solutions of Biolacta N5were prepared in McIlvaines buffer of pH 6.0. The stock solution of-galactosidase from A. oryzae was prepared by adding 37 mg of solidenzyme preparation into 50 mL of McIlvaines buffer of pH 6.0, andthe stock solution of -galactosidase from K. lactis was prepared byadding 0.52 g of liquid enzyme preparation into 4.6 mL of McIlvainesbuffer of pH 8.0.

An Eppendorf tube with a mixture of 790 L of 0.25% (w/w)oNPG solution and 189 L of carbohydrate solution was preheated inan Eppendorf Thermomixer at 40 C and 600 rpm for 10 min.Subsequently, 21 L of enzyme solution was added, and the mixtureswere incubated for another 10 min at 40 C and 600 rpm. In controlmeasurements, enzyme solution was replaced with buffer solution.

A volume of 1.0 mL of 10% (w/w) Na2CO3solution was added tostop the reaction; afterward, the absorbance ofoNP was measured at420 nm. The oNP concentration was determined using the law ofLambertBeer (= 4576 M1cm1). The carbohydrates did not affectthe extinction coefficient. The oNP formation was found to be linearduring the rst 10 min of the reaction. This initial rate of oNPformation was expressed in mmol min1g protein1 unless otherwisestated.

Kinetics of oNPG Conversion in the Presence of Carbohy-drates.In order to determine the Km and vmaxfor oNPG conversionwith Biolacta N5 in the absence and presence of several carbohydrates,oNPG solutions with concentrations varying from 3.4 to 67 mM and aBiolacta N5 solution of approximately 200 mgL1 were prepared. In

addition, solutions were prepared with varying concentrations ofglucose, sucrose, trehalose, raffinose, and dextran-6000 in buffer.During the activity assay, 790 L of oNPG solution, 189 L ofcarbohydrate solution, and 21 L of enzyme solution were incubatedtogether for 0, 3, 5, and 10 min.

A volume of 1.0 mL of 10% (w/w) Na2CO3solution was added tostop the reaction, and, afterward, the absorbance ofoNP was measuredat 420 nm. The initial rate ofoNP formation was determined for eachcombination of initial oNPG concentration and carbohydrateconcentration. The kinetic parameters Km and vmax were determinedwith simple MichaelisMenten kinetics:

=

+

vv S

K S

[ ]

[ ]1

max 1

m 1 (1)

wherev1is the initial reaction rate ofo-nitrophenol formation in mmolmin1g protein1,vmaxis the maximum reaction rate ofoNP formationin mmolmin1g protein1, [S1] is the initial concentration ofoNPGduring the assay in molL1, and Km is the MichaelisMentenparameter for oNPG in molL1.

Water Activity of Carbohydrate Solutions.The water activitiesof solutions of sucrose, trehalose, dextran-6000, and dextran-70 000 inMcIlvaines buffer of pH 6.0 at varying concentrations were measuredwith an Aqualab water activity meter (model 3TE, Decagon Devices,Inc., Pullman, WA, USA) at 40 C. This water activity meter allowsmeasurements from 15 to 40 C with an accuracy of 0.003 aw.Before the water activity was measured, the solutions were kept in awater bath to be equilibrated at 40 C. The measured carbohydrateconcentrations were between 0 and 129 gL1.

Solubility ofoNPG in Carbohydrate Solutions. The solubilityofoNPG in several carbohydrate solutions was determined at 23 or 40C. Solutions of sucrose, trehalose, and raffinose with concentrationsvarying between 4.2 and 33 gL1 were prepared. Subsequently, either0.02 or 0.03 g of oNPG, for the solubility test at 23 or 40 C,respectively, was dissolved in 1.0 mL of the carbohydrate solutions at a

temperature of 65

C. Afterward, the temperature was cooled to 23 or40 C and was constant during the night. During this, crystallizationoccurred.

The obtained suspensions were centrifuged in a Beckman CoulterAllegra X-22R centrifuge for 15 min at 15 500 rpm and 23 or 40 C toremove the crystals from the supernatant. The supernatant wastransferred to cuvettes, and the absorbance was measured with aspectrophotometer at 320 nm. The maximum concentration ofoNPGthat was soluble in the solutions was determined using the law ofLambertBeer. The extinction coefficient was determined to be 2232M1cm1.

Sample Preparation for Analysis of the Product Composi-tion.Volumes of 790 L of 0.25% (w/w)oNPG solution, 189 L of105 gL1 galactose or 108 gL1 glucose or 465 gL1 trehalosesolution, and 21 L of Biolacta N5 (200 mg solidsL1) were

Figure 1.InitialoNPG converting activity of Biolacta N5 as a function of the carbohydrate concentration at 40 C and pH 6.0 in the presence of (A)inert carbohydrates (with sarcosine and dextran-70 000 enzyme concentration during assay = 1.6 mg proteinL1; with dextran-6000, 0.8 mgproteinL1); (B) small carbohydrates (enzyme concentration during assay = 0.8 mg protein L1); and (C) reactants (enzyme concentration duringassay = 2.8 mg proteinL1). The activity is relative to the activity without added carbohydrates (lines for guidance).

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf4008554|J. Agric. Food Chem.2013, 61, 645864646459


3/7

incubated together at 40 C. After 0, 10, 60, and 120 min ofincubation, samples of 200 L were taken and added to 100 L of 5%(w/w) sulfuric acid to inactivate the enzyme. The same procedure wasalso carried out with reference samples in which one or more of thecomponents were replaced with buffer solution.

Before the HPLC analysis, the enzyme was removed from thesamples byltering the samples at 14000gand 18 C for 30 min usingpretreated Amicon Ultra-0.5 centrifugal lter devices (MilliporeCorporation, Billerica, MA, USA) with a cutoffvalue of 10 kDa in aBeckman Coulter Allegra X-22R centrifuge. The pretreatment of thelters consisted of two centrifugation steps: rst, 500 L of Milli-Q water was centrifuged at 14000gat 18 C for 15 min, and second, thelters were placed upside down in the tube and centrifuged at 14000gat 18 C for 5 min. After ltration, the samples were neutralized with5% (w/w) sodium hydroxide.

Measurement of the Carbohydrate Composition.The lteredsamples were analyzed with HPLC using a Rezex RSO oligosaccharide

column (Phenomenex, Amstelveen, The Netherlands) at 80

C. Thecolumn was eluted with Milli-Q water at a ow rate of 0.3 mL/min.The eluent was monitored with a refractive index detector.

The standards that were used for calibration of the column werelactose, glucose, galactose, maltotriose, maltotetraose, maltopentaose,maltohexaose, and maltoheptaose. Galacto-oligosaccharides up to adegree of polymerization of 7 were assumed to have the same responseas the glucose-oligomers with an equal degree of polymerization. Thiswas conrmed with mass balances.

RESULTS AND DISCUSSION

Figure1shows theoNPG converting activity of Biolacta N5 inthe presence of several carbohydrates at varying concentrations.The activity was not greatly affected by the presence of

sarcosine and dextran 70 000, over all tested concentrations.

The activity in the presence of dextran-6000 was slightly higherthan in absence of dextran-6000, but was 60% lower at adextran-6000 concentration of 80 gL1.

In the presence of sucrose, trehalose, and raffinose, theenzyme activity was much higher than without any additives:the activities increased up to 350%. This activity increase issimilar to the activity increase of-amylaseby addition of a.o.sucrose and trehalose described by Yadav.16 In the presence oflactose and Vivinal GOS, the oNPG converting activitydecreased with increasing lactose or Vivinal GOS concen-tration. This may be due to the competition between oNPGand lactose or the oligosaccharides present in Vivinal GOS,

because both are substrates for the enzyme.The products galactose and glucose have a strong positive

effect on the oNPG converting activity: the activities increasedup to 300% and 200%, respectively. Remarkable is the highactivity at low galactose concentrations and in the presence ofglucose. Galactose and glucose are usually recognized to beinhibitors for -galactosidases,1720 but galactose and glucosehere strongly promoted the conversion ofoNPG into oNP. Atgalactose concentrations above 30 gL1, the activity decreasedagain with increasing galactose concentration, which might be asign of dominance of inhibition.

Kinetics of oNPG Conversion in the Presence ofCarbohydrates. The initial rates of oNP formation in thepresence of glucose, sucrose, trehalose, raffinose, and dextran-6000 were measured (see Figure S1 in the Supporting

Figure 2. MichaelisMenten constant Km of -galactosidase from Bacillus circulans for oNPG plotted as a function of the carbohydrateconcentration, in (A) mM and (B) gL1, at 40 C and pH 6.0 (lines for guidance).

Figure 3. Maximum reaction rate of oNP formation vmaxwith -galactosidase from Bacillus circulans plotted as a function of the carbohydrateconcentration, in (A) mM and (B) gL1, at 40 C and pH 6.0 (lines for guidance).




4/7

Information). The data were tted to the MichaelisMentenmodel to obtain the kinetic parameters Kmand vmax, which areshown in Figures2 and 3, respectively.

Figure 2 shows that the MichaelisMenten constant Kmdecreased with the addition of carbohydrates. On a molarconcentration scale, this decrease was more or less independentof the type of carbohydrate added. However, on a massconcentration scale, dextran affected the K

mless than smaller

carbohydrates, due to its larger molecular weight. The smallercarbohydrates affected the Km all in a similar way.

Below carbohydrate concentrations of approximately 300mM, the Km decreased with increasing carbohydrate concen-tration, which implies that the affinity of the enzyme for thesubstrate oNPG increased with the addition of thesecarbohydrates. At higher carbohydrate concentrations, the Kmincreased again slightly with further increase of the carbohy-drate concentration.

Figure3shows more complex effects onvmaxfor each of thetested carbohydrates (both on a molar concentration scale andon a mass concentration scale).vmaxdid not change signicantly

with the glucose concentration, but in the presence of sucrose,trehalose, and raffinose, the vmax increased strongly withincreasing carbohydrate concentration, with the largest effectfor raffinose and the smallest for sucrose. Dextran-6000 had anegative effect on vmax: it decreased with increasing dextran-6000 concentration.

Characteristics of Reaction Medium. To exclude effectsby the medium from causing changes in enzyme activity andenzyme kinetics with the addition of carbohydrates, the wateractivity and the solubility of oNPG in the carbohydratesolutions were measured.

Water Activity.The water activities of solutions of sucrose,trehalose, dextran-6000, and dextran-70 000 in buffer weremeasured at 40 C (see Figure S2 in the SupportingInformation). When no carbohydrates were present, the

water activity was approximately 0.994. The fact that the

water activity is not 1 at carbohydrate concentration 0 is due tothe presence of the buffer. The water activities of each of thecarbohydrate solutions were found to show only a very slightdecrease from approximately 0.994 to 0.986 over a concen-tration range up to 130 gL1. This small decrease cannotexplain the large changes in enzyme activity and enzymekinetics. Gosling et al.21 found earlier that oligosaccharide levels

were not affected by even larger changes in water activity.Solubility of oNPG in Carbohydrate Solutions. The

addition of carbohydrates could lead to a shift in thedistribution ofoNPG molecules between being free in solutionand being bound to the enzyme, since the associated state willhave a lower overall volume than the dissociated state. That

would mean that with the addition of carbohydrates theoNPG

molecule prefers to leave the crowded environment and prefersto bind to the enzyme, which could result in a decrease in Kmand an increase in the enzyme activity. If this is the case, thesolubility of oNPG is expected to decrease with addition ofcarbohydrates when no enzyme is present. The solubility ofoNPG in buffer was determined to be 15 1 and 21 0 gL1

at 23 and 40 C, respectively (see Figure S3 in the SupportingInformation). The addition of sucrose, trehalose, and raffinosein the tested concentration ranges did not change the solubility,

which means that the changes in enzyme activity and enzymekinetics were not caused by a change in the solubility ofoNPG.

Since the water activity and the solubility ofoNPG do notchange with increasing carbohydrate concentration, the

characteristics of the reaction medium can be excluded ascause of the changes in enzyme activity and enzyme kinetics.

Characteristics of the Enzyme. Since the characteristicsof the reaction medium do not change with the addition of thecarbohydrates, we also investigated the effect o f thecarbohydrates on the enzyme itself.

In general, oNPG conversion with -galactosidases takesplace in two steps. First, an enzymegalactose complex isformed and oNP is released, and second, the galactose isreleased since water is used as the acceptor molecule; hydrolysistakes place. However, in the presence of carbohydrates,transglycosylation might take place. The reaction mechanismthat we propose is shown in Figure 4. Instead of water, the

carbohydrates may also be used as an acceptor molecule andform a product together with the galactose unit that is releasedfrom the enzymegalactose complex. A similar phenomenon

was observed by Gosling et al.21 during lactose conversion inthe presence of sucrose. In this case, the product should have adegree of polymerization that is larger than the carbohydrateadded. To test this hypothesis, we measured the productcomposition during oNPG conversion with Biolacta N5 inpresence of galactose, glucose, and trehalose. The results areshown in Figure5.

During the conversion of oNPG without any addedcarbohydrates, a slight decrease in oNPG concentration and aslight increase in galactose concentration were detected. Whengalactose, glucose, or trehalose was added, much more oNPG

was converted, which is in agreement with the increases inactivity (Figure1). In addition, disaccharides were formed withaddition of galactose and glucose, whereas trisaccharides wereformed in the presence of trehalose. This conrms ourhypothesis that the added carbohydrates participate in thereaction, and removing galactose from the active center, freesup the enzyme for faster oNPG conversion.

Some carbohydrates that do not contain a galactose moietycan act only as acceptor and thus will speed up the overall

reaction by freeing up the active center; other sugars (such asgalactose itself) may also act as donor, and in this case bothcompetitive inhibition and the acceptor-acceleration take place.In this case, one may expect the optimum as was observed inFigure1C.

Changes in Km and vmax Explained. The increase inaffinity caused by the addition of carbohydrates may beexplained by molecular crowding. A similar Km decrease wasreported before by Jiang andGuo22 for addition of dextrans toisochorismate synthase. Yadav16 described an activity increaseof-amylase by addition of a.o. sucrose and trehalose, which

was due to molecular crowding. The association of the enzymewith oNPG probably results in a volume reduction, which is

Figure 4. Schematic representation of the reaction mechanism of-

galactosidase fromBacillus circulans. Abbreviations: E, enzyme; oNPG,o-nitrophenyl -D-galactopyranoside; oNP, o-nitrophenol; E-gal,enzymegalactose complex; gal, galactose; carb, carbohydrate; carb-gal, oligosaccharide that consists of one galactose unit attached toanother carbohydrate.




5/7

thermodynamically favorable in a crowded environment.10,23

Besides, if this enzyme is active as a folded protein, crowdingwill stabilize the folded proteins.10,23

The smaller carbohydrates have a stronger effect on a mass

concentration scale on theKmthan dextran, which is inert. Thismay be explained by dextran having a smaller total excludedvolume per gram than the small carbohydrates, although thetotal volume of the carbohydrates is equal in each case. Figure 6shows the calculated total excluded volume for various

concentrations of added carbohydrates. The calculations werecarried out assuming the carbohydrate and the substrate to bespheres with radiiRcandRs, withR= 8.2610

1MW1/3 nm

(MW in kD), similar to the approach by Batra et al.24 for

dextran. The total excluded volume is equal to the number ofcarbohydrate molecules multiplied by the excluded volume ofeach carbohydrate molecule with radius Rc + Rs, since thecenter of the substrate cannot enter there. This results in alarger total excluded volume when a certain mass of smallcarbohydrates is present comparedto when the same mass oflarge carbohydrates is present.11,12 Therefore, the effect ofmolecular crowding in the case of small carbohydrates on the

Km is larger than in the case of dextran. In addition, theinteractions of the small carbohydrates might also play a role inthe reaction.

The slight increase of Km at higher carbohydrate concen-trations might be a result of diffusion limitation, becausediffusion is slower in crowded systems, and thus the frequency

o f the encounters between enzyme and substrate isreduced.10,23

The maximum rate of oNP formation (vmax) was higher inthe presence of sucrose, trehalose, or raffinose than withdextran mainly because these carbohydrates are acceptors in thereaction. They cause a quick release of the enzyme (from theenzymegalactose complex), and the enzyme is free in solutionagain, so that anotheroNPG molecule can form a complex withthe enzyme. Dextran is inert and cannot be used as an acceptormolecule.

Since an increase in vmax was found with increasingcarbohydrate concentration, the attachment of an acceptormolecule (either water or a carbohydrate molecule) is supposed

Figure 5. oNPG and carbohydrate proles during oNPG conversion at 2.0 gL1 with 0.80.9 mgL1 Biolacta N5 at 40 C and pH 6.0 in thepresence of (A) no added carbohydrates, (B) 20 gL1 galactose, (C) 20 gL1 glucose, and (D) 88 gL1 trehalose. (The reaction was followed fortwo hours (whereas the activity assay is 10 min) to nd larger differences in breakdown or formation of the components.)

Figure 6.Calculated total excluded volume for substrate molecules asa function of the carbohydrate concentration. The total excludedvolume is the amount of carbohydrate molecules multiplied with theexcluded volume of each carbohydrate molecule. Carbohydrate andsubstrates are assumed to be spherical molecules with radii Rcand Rs,with R= 8.26 101 MW1/3 nm (MW in kD). The excludedvolume of each carbohydrate molecule has a radius ofRc+ Rs.




6/7

to be the rate-limiting step. The reaction is accelerated whenthe carbohydrate concentration is increased. This implies that

water is a very poor acceptor molecule, as the molar water

content is still very high compared to the carbohydrateconcentration and water molecules are very mobile. Theaffinity of the enzymegalactose complex for the speciccarbohydrate molecules therefore has to be much higher thanthe affinity for a water molecule.

The addition of glucose does not changevmax. According toFigure 5, the lowest conversion of oNPG was found withglucose among the tested carbohydrates. This can beexplained

by glucose acting as an uncompetitive inhibitor.18,25 If glucoseinhibits the reaction while also acting as an acceptor, these twoeffects may cancel out each other.Next to glucose, sucrose isalso an uncompetitive inhibitor.25 However, the inhibitionconstant of sucrose is higher than that of glucose,25 whichmeans that the affinity for sucrose (as an inhibitor) is lower

than for glucose. The inhibiting eff

ect can explain the vmaxcaused by glucose being lower than that of sucrose. Raffinoseand trehalose do not have any inhibiting effect.

The presence of dextran decreased vmax at high concen-trations, which may be due to diffusion limitations.

Overall, the effect of carbohydrates acting in the reaction asacceptors, represented by a change in vmax, is large compared tothe effect of molecular crowding on the enzyme activity,represented by a decrease in Km.

Mechanistic Understanding of-Galactosidase Activ-ity.For a better mechanistic understanding of the activity of-galactosidases, theoNPG converting activity of-galactosidasesofA. oryzaeand K. lactiswas measured in the presence of smallcarbohydrates. The results are shown in Figure 7.

Figure7 shows, together with Figure1C, that the reactants(lactose, oligosaccharides, galactose, and glucose) affect thethree enzymes in very different ways. In production systems

with -galactosidase of B. circulans with a high lactoseconcentration, where lactose is converted into GOS, thecompetition between lactose and Vivinal GOS for association

with the enzyme is advantageous: the enzyme has a high affinityfor these components and will form products with a higherdegree of polymerization. The latter was previously observed byBoon et al.6 The decrease in activity in the presence ofgalactose does not hinder the reaction, since hydrolysis islimited in concentrated solutions, and thus the galactoseconcentration remains low.

TheoNPG converting activity of-galactosidase ofA. oryzae(Figure7A) is (slightly) higher in the presence of lactose andglucose, but lower in the presence of Vivinal GOS and

galactose, with the lowest activity in the presence of galactose.The higher activity in the presence of lactose implies thatlactose is used as an acceptor molecule instead of a donormolecule: the affinity of the enzyme for oNPG is much higherthan for lactose, which will not be able to displace oNPG. Weexpect glucose to act as an acceptor as well. Galactose inhibitsthe enzyme already at low concentrations, which is inagreement with the conclusion by Boon et al.6 that galactoseis an inhibitor.

The oNPG converting activity of-galactosidase ofK. lactis(Figure7B) showed similar trends. Vivinal GOS reduced theactivity. Lactose does not affect the activity much, but itprobably acts simultaneously as an acceptor and as a donor. As

with-galactosidase fromA. oryzae, glucosewill be an acceptor;galactose once more acts as an inhibitor.6 Since this enzymeshows much more hydrolysis activity, product inhibition bygalactose would probably hinder the production of GOS evenat high lactose concentrations.

Previously it was shown that Biolacta N5 was a productiveenzyme preparation in terms of oligosaccharide yield comparedto other -galactosidases.6,7 This can now be explained by themuch higher affinity for lactose and the suitability of glucoseand galactose as acceptor molecules. Besides, galactose is notinhibiting the enzyme that strongly. These mechanisticdifferences are the reason that the enzyme from B. circulansshows much higher production rates of GOS than the -galactosidases of A. oryzae and K. lactis under comparableconditions.

ASSOCIATED CONTENT*S Supporting Information

Figure S1: Measured initial rates of oNP formation in thepresence of various carbohydrates including their t withMichaelisMenten kinetics. Figure S2: Water activity of variouscarbohydrate solutions. Figure S3: Solubility of oNPG in

various carbohydrate solutions. This material is available free ofcharge via the Internet athttp://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Tel: +31 317 482231. E-mail:[email protected].

Figure 7.InitialoNPG converting activity of-galactosidase from (A)Aspergillus oryzaeat 15 mg solidsL1, 40 C, and pH 6.0 and (B)Kluveromyceslactisat 144 mg enzyme preparationL1, 20 C, and pH 8.0, as a function of the carbohydrate concentration in presence of various reactants (linesfor guidance).


http://pubs.acs.org/mailto:[email protected]:[email protected]://pubs.acs.org/


7/7

Funding

This project is jointly nanced by the European Union,European Regional Development Fund and The Ministry ofEconomic Affairs, Agriculture and Innovation, Peaks in theDelta, the Municipality of Groningen, the Provinces ofGroningen, Fryslan, and Drenthe, and the Dutch CarbohydrateCompetence Center (CCC WP9).

NotesThe authors declare no competing nancial interest.

ACKNOWLEDGMENTS

The authors would like to thank Wanqing Jia for her help withthe activity measurements and Eric Benjamins, Linqiu Cao,Ellen van Leusen, Albert van der Padt, and Jan Swarts ofFrieslandCampina for the valuable scientic discussions.

ABBREVIATIONS USED

GOS, galacto-oligosaccharides; oNPG, o-nitrophenyl -D-galactopyranoside;oNP, o-nitrophenol

REFERENCES(1) Barreteau, H.; Delattre, C.; Michaud, P. Production of

oligosaccharides as promising new food additive generation. FoodTechnol. Biotechnol. 2006, 44, 323333.

(2) Macfarlane, G. T.; Steed, H.; Macfarlane, S. Bacterial metabolismand health-related effects of galacto-oligosaccharides and otherprebiotics. J. Appl. Microbiol. 2008, 104, 305344.

(3) Playne, M. J.; Crittenden, R. G. Galacto-oligosaccharides andother products derived from lactose. In Advanced Dairy Chemistry;McSweeney, P. L. H.; Fox, P. F., Eds.; Springer: New York, 2009; Vol.3, pp 121201.

(4) Mahoney, R. R. Galactosyl-oligosaccharide formation duringlactose hydrolysis: a review. Food Chem. 1998, 63, 147154.

(5) Prenosil, J. E.; Stuker, E.; Bourne, J. R. Formation ofoligosaccharides during enzymatic lactose: part I: state of art.

Biotechnol. Bioeng. 1987, 30, 10191025.

(6) Boon, M. A.; Janssen, A. E. M.; vant Riet, K. Effect oftemperature and enzyme origin on the enzymatic synthesis ofoligosaccharides.Enzyme Microb. Technol. 2000, 26, 271281.

(7) Nakanishi, K.; Matsuno, R.; Torii, K.; Yamamoto, K.; Kamikubo,T. Properties of immobilized -galactosidase from Bacillus circulans.

Enzyme Microb. Technol.1983, 5, 115120.(8) Urrutia, P.; Rodriguez-Colinas, B.; Fernandez-Arrojo, L.;

Ballesteros, A. O.; Wilson, L.; Illanes, A.; Plou, F. J. Detailed analysisof galactooligosaccharides synthesis with -galactosidase from

Aspergillus oryzae. J. Agric. Food Chem. 2013, 61, 10811087.(9) Huber, R. E.; Kurz, G.; Wallenfels, K. A quantitation of the

factors which affect the hydrolase and transgalactosylase activities of-galactosidase (E. coli) on lactose. Biochemistry 1976, 15, 19942001.

(10) Ellis, R. J. Macromolecular crowding: obvious but under-appreciated.Trends Biochem. Sci. 2001, 26, 597604.

(11) Minton, A. P. The influence of macromolecular crowding andmacromolecular confinement on biochemical reactions in physio-logical media. J. Biol. Chem. 2001, 276, 1057710580.

(12) Zhou, H. X.; Rivas, G. N.; Minton, A. P. Macromolecularcrowding and connement: biochemical, biophysical, and potentialphysiological consequences. In Annual Review of Biophysics; AnnualReviews: Palo Alto, CA, 2008; Vol. 37, pp 375397.

(13) Zimmerman, S. B.; Minton, A. P. Macromolecular crowding:biochemical, biophysical, and physiological consequences. Annu. Rev.Biophys. Biomol. Struct.1993, 22, 2765.

(14) Kim, J. S.; Yethiraj, A. Effect of macromolecular crowding onreaction rates: a computational and theoretical study. Biophys. J.2009,96, 13331340.

(15) Warmerdam, A.; Paudel, E.; Jia, W.; Janssen, A. E. M.; Boom, R.M. Characterization of-galactosidase isoforms from Bacillus circulans

and their contribution to GOS production. Appl. Biochem. Biotechnol.2013, 170 (2), 340358.

(16) Yadav, J. K. Macromolecular crowding enhances catalyticefficiency and stability of-amylase. ISRN Biotechnol. 2013, 2013.

(17) Bakken, A. P.; Hill, C. G., Jr.; Amundson, C. H. Hydrolysis oflactose in skim milk by immobilized -galactosidase (Bacilluscirculans). Biotechnol. Bioeng. 1992, 39, 408417.

(18) Boon, M. A.; Janssen, A. E. M.; van der Padt, A. Modelling and

parameter estimation of the enzymatic synthesis of oligosaccharides by-galactosidase from Bacillus circulans. Biotechnol. Bioeng. 1999, 64,558567.

(19) Chockchaisawasdee, S.; Athanasopoulos, V. I.; Niranjan, K.;Rastall, R. A. Synthesis of galacto-oligosaccharide from lactose using -galactosidase from Kluyveromyces lactis: studies on batch andcontinuous UF membrane-fitted bioreactors. Biotechnol. Bioeng.2005, 89, 434443.

(20) Neri, D. F. M.; Balcao, V. M.; Costa, R. S.; Rocha, I. C. A. P.;Ferreira, E. M. F. C.; Torres, D. P. M.; Rodrigues, L. R. M.; Carvalho,L. B., Jr.; Teixeira, J. A. Galacto-oligosaccharides production duringlactose hydrolysis by free Aspergillus oryzae -galactosidase andimmobilized on magnetic polysiloxane-polyvinyl alcohol. Food Chem.2009, 115, 9299.

(21) Gosling, A.; Stevens, G. W.; Barber, A. R.; Kentish, S. E.; Gras,S. L. Effect of the substrate concentration and water activity on the

yield and rate of the transfer reaction of-galactosidase from Bacilluscirculans. J. Agric. Food Chem. 2011, 59, 33663372.

(22) Jiang, M.; Guo, Z. Effects of macromolecular crowding on theintrinsic catalytic efficiency and structure of enterobactin-specificisochorismate synthase. J. Am. Chem. Soc. 2007, 129, 730731.

(23) van Boekel, M. A. J. S. Kinetic Modeling of Reactions in Foods;CRC Press: Wageningen University, The Netherlands, 2009; p 767.

(24) Batra, J.; Xu, K.; Qin, S.; Zhou, H.-X. Effect of macromolecularcrowding on protein binding stability: modest stabilization andsignificant biological consequences. Biophys. J. 2009, 97, 906911.

(25) Deschavanne, P. J.; Viratelle, O. M.; Yon, J. M. Conformationaladaptability of the active site of beta-galactosidase. Interaction of theenzyme with some substrate analogous effectors. J. Biol. Chem. 1978,253, 833837.



onpg beta lactase

Documents